The hydrolysis of chemical bonds within molecules is of critical importance in most metabolic (e.g., catabolic and anabolic) pathways in cells. A large family of enzymes which catalyze the cleavage of a bond with the addition of water, termed hydrolases, has been identified. Members of the hydrolase family are found in nearly all organisms, from microbes to plants to humans. Different classes of hydrolases are specific for an array of biological and chemical substrates. Members of the hydrolase family of enzymes include enzymes that hydrolyze ester bonds (e.g., phosphatases, sulfatases, exonucleases, and endonucleases), glycosidases, enzymes that act on ether bonds, peptidases (e.g., exopeptidases and endopeptidases), as well as enzymes that hydrolyze carbon-nitrogen bonds, acid anhydrides, carbon-carbon bonds, halide bonds, phosphorous-nitrogen bonds, sulfur-nitrogen bonds, carbon-phosphorous bonds, and sulfur-sulfur bonds (E. C. Webb ed., Enzyme Nomenclature, pp. 306-450, (copyright)1992 Academic Press, Inc. San Diego, Calif.).
Hydrolases vary widely in primary sequence, substrate specificity, and physical properties. However, despite the lack of sequence homology, hydrolase family members display structural similarities, e.g., conservation of a catalytic site framework. For example, the alpha/beta hydrolase fold is a structural motif that is common to a variety of hydrolytic enzymes including, lipases, e.g., fungal, bacterial and pancreatic lipase, acetylcholinesterases, serine carboxypeptidases, haloalkane dehalogenases, dienelactone hydrolases, A2 bromoperoxidases, and thioesterases (Schrag, J. et al. (1997) Meth. Enzymol. 284:85-107). Enzymes possessing the alpha/beta hydrolase fold have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues (Ollis, D. et al. (1992) Protein Eng. 5:197-211). In particular, one conserved feature of the alpha/beta hydrolase fold is a nucleophile-histidine-acid catalytic triad. The identities of the triad residues in alpha/beta hydrolase fold enzymes are quite variable in that serine, aspartate, and cysteine have all been identified as catalytic nucleophiles (Schrag, J. et al. supra).
Hydrolases play important roles in the synthesis and breakdown of nearly all major metabolic intermediates, including polypeptides, nucleic acids, and lipids. As such, their activity contributes to the ability of the cell to grow and differentiate, to proliferate, to adhere and move, and to interact and communicate with other cells. Hydrolases also are important in the conversion of pro-proteins and pro-hormones to their active forms, the inactivation of peptides, the biotransformation of compounds (e.g., a toxin or carcinogen), antigen presentation, and the regulation of synaptic transmission.
The present invention is based, at least in part, on the discovery of novel members of the family of hydrolase molecules, referred to herein as xe2x80x9chydrolase-1xe2x80x9d or xe2x80x9cHYDL-1xe2x80x9d nucleic acid and protein molecules. The HYDL-1 nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, differentiation, or migration. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding HYDL-1 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of HYDL-1-encoding nucleic acids.
In one embodiment, an HYDL-1 nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 74%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1 or 3.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-46 of SEQ ID NO:1. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 995-1332 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 1 or 3.
In another embodiment, an HYDL-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, an HYDL-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 74%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human HYDL- 1. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length and encodes a protein having an HYDL-1 activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably HYDL-1 nucleic acid molecules, which specifically detect HYDL-1 nucleic acid molecules relative to nucleic acid molecules encoding non-HYDL-1 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1 or 3.
In preferred embodiments, the nucleic acid molecules are at least 15 nucleotides (e.g., 15 contiguous nucleotides) in length and hybridize under stringent conditions to the nucleotide molecules set forth in SEQ ID NO:1 or 3 or a complement thereof. In certain embodiments, the nucleic acid molecules are at least 15 nucleotides in length and hybridize under stringent conditions to nucleotides 1-23 and 1002-1332 of SEQ ID NO:1. In another embodiment, the nucleic acid molecules comprise nucleotides 1-23 and 1002-1332 of SEQ ID NO:1. In yet another embodiment, the nucleic acid molecules consist of nucleotides 1-23 and 1002-1332 of SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2., wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a HYDL-1 nucleic acid molecule, e.g., the coding strand of an HYDL-1 nucleic acid molecule.
Another aspect of the invention provides a vector comprising an HYDL-1 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably an HYDL-1 protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant HYDL-1 proteins and polypeptides. In one embodiment, an isolated HYDL-1 protein includes at least one or more of the following domains: an alpha/beta hydrolase fold, a transmembrane domain, or a signal peptide. In a preferred embodiment, an isolated HYDL-1 protein includes at least one alpha/beta hydrolase fold.
In a preferred embodiment, an HYDL-1 protein includes at least one or more of the following domains: an alpha/beta hydrolase fold, a transmembrane domain, or a signal peptide, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 72%, 75%, 80%, 85%, 87%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, an HYDL-1 protein includes at least one or more of the following domains: an alpha/beta hydrolase fold, a transmembrane domain, or a signal peptide, and has an HYDL-1 activity (as described herein).
In yet another preferred embodiment, an HYDL-1 protein includes at least one or more of the following domains: an alpha/beta hydrolase fold, a transmembrane domain, or a signal peptide, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.
In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2. In another embodiment, an HYDL-1 protein has the amino acid sequence of SEQ ID NO:2.
In another embodiment, the invention features an HYDL-1 protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 72%, 75%, 80%, 85%, 87%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof. This invention further features an HYDL-1 protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-HYDL-1 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably HYDL-1 proteins. In addition, the HYDL-1 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of an HYDL-1 nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an HYDL-1 nucleic acid molecule, protein, or polypeptide such that the presence of an HYDL-1 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of HYDL-1 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of HYDL-1 activity such that the presence of HYDL-1 activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating HYDL-1 activity comprising contacting a cell capable of expressing HYDL-1 with an agent that modulates HYDL-1 activity such that HYDL-1 activity in the cell is modulated. In one embodiment, the agent inhibits HYDL-1 activity. In another embodiment, the agent stimulates HYDL-1 activity. In one embodiment, the agent is an antibody that specifically binds to an HYDL-1 protein. In another embodiment, the agent modulates expression of HYDL-1 by modulating transcription of an HYDL-1 gene or translation of an HYDL-1 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an HYDL-1 mRNA or a HYDL-1 gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted HYDL-1 protein or nucleic acid expression or activity by administering an agent which is an HYDL-1 modulator to the subject. In one embodiment, the HYDL-1 modulator is an HYDL-1 protein. In another embodiment the HYDL-1 modulator is an HYDL-1 nucleic acid molecule. In yet another embodiment, the HYDL-1 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant or unwanted HYDL-1 protein or nucleic acid expression is a hydrolase-associated disorder, e.g., a central nervous system (CNS) disorder, a cardiovascular disorder, a muscular disorder, a hormonal disorder, a gastrointestinal disorder, a metabolic disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, or migration disorder. In another preferred embodiment, the disorder characterized by aberrant or unwanted HYDL-1 protein or nucleic acid expression is a cancer, e.g., lung cancer.
The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an HYDL-1 protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of an HYDL-1 protein, wherein a wild-type form of the gene encodes a protein with an HYDL-1 activity.
In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of an HYDL-1 protein, by providing an indicator composition comprising an HYDL-1 protein having HYDL-1 activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on HYDL-1 activity in the indicator composition to identify a compound that modulates the activity of a HYDL-1 protein.
Accordingly, the present invention provides methods and compositions for the diagnosis and treatment of cellular proliferative disorders, including but not limited to cancer, e.g., lung, colon and breast cancer.
Other features and advantages of the invention will be apparent from the following detailed description and claims.